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Two different antibodies necessary for sandwich ELISA? - (Aug/22/2006 )

Is it necessary to have antibodies with two different specificities for a sandwich ELISA, or is it possible, for instance, to use the same polyclonal or monoclonal antibody for both parts of the sandwich. I have only one polyclonal antibody available for an equine cytokine of interest. Thank you in advance...


you should be able to use the polyclonal ab for a sandwich elisa, although it may not be as sensitive.



the question in this case is how do you specifically detect the second Ab? You will get a high background due to the capture Ab... If the background is too high compared to the signal of the second layer, i suggest you to crosslink a biotin on some of your pAb and reveal it with (S)Av coupled to an enzyme


Why not to use the same monoclonal antibody for ELISA : because if your antigen has only one epitope per molecule, the second antibody will never be able to bind, because the first is already bound to the unique epitope.
If you use a polyclonal antibody, there is a better chance that there are several epitopes per molecule and that not all epitopes will be bound by the first antibody. Not sure.
you have to try.

Of course, as said tryptofan, you can't use an anti immunoglobulin to detect the antibody. You need to label your second antibody with biotin for example, and detect with streptavidin-HRP.